The agent mainly responsible for fibrinolysis is plasmin, the activated form of plasminogen. Many substances can activate plasminogen, including activated Hageman factor, streptokinase, urokinase (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (pKA). pKA is both an activator of the zymogen form of urokinase and a direct plasminogen activator.
Plasmin is undetectable in normal circulating blood, but plasminogen, the zymogen, is present at about 3 μM. An additional, unmeasured amount of plasminogen is bound to fibrin and other components of the extracellular matrix and cell surfaces. Normal blood contains the physiological inhibitor of plasmin, α2-plasmin inhibitor (α2-PI), at about 2 μM. Plasmin and α2-PI form a 1:1 complex. Matrix or cell bound-plasmin is relatively inaccessible to inhibition by α2-PI. Thus, activation of plasmin can exceed the neutralizing capacity of α2-PI causing a profibrinolytic state.
Plasmin, once formed, degrades fibrin clots, sometimes prematurely; digests fibrinogen (the building material of clots) impairing hemostasis by causing formation of friable, easily lysed clots from the degradation products, and inhibition of platelet adhesion/aggregation by the fibrinogen degradation products; interacts directly with platelets to cleave glycoproteins Ib and IIb/IIIa preventing adhesion to injured endothelium in areas of high shear blood flow and impairing the aggregation response needed for platelet plug formation (ADEL86); proteolytically inactivates enzymes in the extrinsic coagulation pathway further promoting a prolytic state.
Inappropriate fibrinolysis and fibrinogenolysis leading to excessive bleeding is a frequent complication of surgical procedures that require extracorporeal circulation, such as cardiopulmonary bypass, and is also encountered in thrombolytic therapy and organ transplantation, particularly liver. Other clinical conditions characterized by high incidence of bleeding diathesis include liver cirrhosis, amyloidosis, acute promyelocytic leukemia, and solid tumors. Restoration of hemostasis requires infusion of plasma and/or plasma products, which risks immunological reaction and exposure to pathogens, e.g. hepatitis virus and HIV.
Very high blood loss can resist resolution even with massive infusion. When judged life-threatening, the hemorrhage is treated with antifibrinolytics such as ε-amino caproic acid (See HOOV93) (EACA), tranexamic acid, or aprotinin (NEUH89). Aprotinin is also known as Trasylolu and as Bovine Pancreatic Trypsin Inhibitor (BPTI). Hereinafter, aprotinin will be referred to as “BPTI.” EACA and tranexamic acid only prevent plasmin from binding fibrin by binding the kringles, thus leaving plasmin as a free protease in plasma. BPTI is a direct inhibitor of plasmin and is the most effective of these agents. Due to the potential for thrombotic complications, renal toxicity and, in the case of BPTI, immunogenicity, these agents are used with caution and usually reserved as a “last resort” (PUTT89). All three of the antifibrinolytic agents lack target specificity and affinity and interact with tissues and organs through uncharacterized metabolic pathways. The large doses required due to low affinity, side effects due to lack of specificity and potential for immune reaction and organ/tissue toxicity augment against use of these antifibrinolytics prophylactically to prevent bleeding or as a routine postoperative therapy to avoid or reduce transfusion therapy. Thus, there is a need for a safe antifibrinolytic.
Excessive bleeding can result from deficient coagulation activity, elevated fibrinolytic activity, or a combination of the two conditions. In most bleeding diatheses one must control the activity of plasmin. The clinically beneficial effect of bovine pancreatic trypsin inhibitor (BPTI) in reducing blood loss is thought to result from its inhibition of plasmin (Kd approximately 0.3 nM) or of plasma kallikrein (Kd approximately 100 nM) or both enzymes.
Interestingly, BPTI-induced hypersensitivity reaction occurs in about 1.2 to 2.7 percent of patients reexposed to aprotinin (30). Of these reactions 50 percent are life threatening with 9 percent fatality rate (30). Thus, a human molecule that is selectively modified to make it more potent is highly desirable. Such molecule is also expected to be less immunogenic. Side effects and toxicity issues for the use of BPTI have recently been outlined (Manago et al., N Engl J Med 2006; 354:353-65). Textilinin has also been compared with aprotinin, however, textilinin is a snake protein and therefore has immunogenecity issues associated with it. (Pathophysiol Haemost Thromb. 2005; 34(4-5):188-93 and U.S. Pat. No. 7,070,969).
What is needed in the art is a plasmin inhibitor that is as potent (or more potent) than BPTI, but that is almost identical to a human protein domain, thereby offering similar therapeutic potential but posing less potential for antigenicity.